The mouse uterus has provided a system for study of estrogen action since it contains estrogen receptors and depends on estrogen stimulation for maintenance of physiological functions. We have previously identified an estrogen-stimulated mouse uterine secretory protein (Mr about 70 x 1000) by in vitro 35S-methionine labeling experiments. Currently, the HPLC purified 70K protein was further characterized. Total amino acid analysis did not show an unusual composition. The 70K protein is a glycoprotein with an apparent asparagine-linked carbohydrate moiety. Individual sugar analysis revealed a single carbohydrate chain which contains sialic acid, galatose, mannose, fucose, and glucosamine. The NH2 terminus of the 70K protein was cleaved with cyanogen bromide, and the resulting fragments were separated by HPLC. Two of the fragments yielded amino acid sequence. We have obtained the 32 mer synthetic oligonucleotides according to the amino acid sequence which will be used as the probe to select clones containing the cDNA insert coding for the 70K protein mRNA. Rabbit polyclonal antibody raised against the purified 70K protein demonstrated specificity for the 70K protein by "Western Blot" analysis. The uterine 70K protein was induced by estrogen but not by testosterone or progesterone. Slot blot analysis and the immuno-enzyme-linked method was used to examine the tissue distribution of the 70K protein. Tissues such as lung, brain, spleen, ovary, kidney, liver, muscle and intestine of estrogen-treated mice did not have measurable amounts of 70K protein. Only uterine and vaginal tissue gave positive reactions.